Biomedical subjects

Frederick S Southwick

Publications and source records attributed to Frederick S Southwick.

9 recordsLinked to original sources

PubMed2006

A CapG gain-of-function mutant reveals critical structural and functional determinants for actin filament severing.

CapG is the only member of the gelsolin family unable to sever actin filaments. Changing amino acids 84-91 (severing domain) and 124-137 (WH2-containing segment) simultaneously to the sequences of gelsolin results in a mutant, CapG-sev, capable of severing actin filaments. The gain of severing function does not alter actin filament capping, but is accompanied by a higher affinity for monomeric actin, and the capacity to bind and sequester two actin monomers. Analysis of CapG-sev crystal structure suggests a more loosely folded inactive conformation than gelsolin, with a shorter S1-S2 latch. Calcium binding to S1 opens this latch and S1 becomes separated from a closely interfaced S2-S3 complex by an extended arm consisting of amino acids 118-137. Modeling with F-actin predicts that the length of this WH2-containing arm is critical for severing function, and the addition of a single amino acid (alanine or histidine) eliminates CapG-sev severing activity, confirming this prediction. We conclude that efficient severing utilizes two actin monomer-binding sites, and that the length of the WH2-containing segment is a critical functional determinant for severing.

Actin Cytoskeleton↗

PubMed2005

Anthrax lethal toxin paralyzes neutrophil actin-based motility.

Bacillus anthracis causes high-level bacteremia, strongly suggesting paralysis of the innate immune system. We have examined the effects of anthrax lethal toxin (LT) on human neutrophil chemotaxis, a process that requires actin filament assembly. Polymorphonuclear neutrophils (PMNs) treated with a sublethal concentration of LT (50 ng/mL) for 2 h demonstrated insignificant apoptosis or necrosis. However, this same concentration slowed human PMN formylmethionylleucylphenylalanine (FMLP)-stimulated chemokinesis by >60%, markedly reduced polar morphology, and rendered PMNs incapable of responding to a chemotactic gradient. These changes were accompanied by a >50% reduction in FMLP-induced actin filament assembly. One hour of exposure to LT failed to impair polarity or actin assembly, and the effects of LT were independent of mitogen-activated protein kinase kinase 1 inhibition. We conclude that 2 h of exposure to LT markedly impairs PMN actin assembly, and reductions in actin filament content are accompanied by a profound paralysis of PMN chemotaxis.

Actins↗

PubMed2003

Depolymerization of actin filaments by profilin. Effects of profilin on capping protein function.

Profilin interacts with the barbed ends of actin filaments and is thought to facilitate in vivo actin polymerization. This conclusion is based primarily on in vitro kinetic experiments using relatively low concentrations of profilin (1-5 microm). However, the cell contains actin regulatory proteins with multiple profilin binding sites that potentially can attract millimolar concentrations of profilin to areas requiring rapid actin filament turnover. We have studied the effects of higher concentrations of profilin (10-100 microm) on actin monomer kinetics at the barbed end. Prior work indicated that profilin might augment actin filament depolymerization in this range of profilin concentration. At barbed-end saturating concentrations (final concentration, approximately 40 microm), profilin accelerated the off-rate of actin monomers by a factor of four to six. Comparable concentrations of latrunculin had no detectable effect on the depolymerization rate, indicating that profilin-mediated acceleration was independent of monomer sequestration. Furthermore, we have found that high concentrations of profilin can successfully compete with CapG for the barbed end and uncap actin filaments, and a simple equilibrium model of competitive binding could explain these effects. In contrast, neither gelsolin nor CapZ could be dissociated from actin filaments under the same conditions. These differences in the ability of profilin to dissociate capping proteins may explain earlier in vivo data showing selective depolymerization of actin filaments after microinjection of profilin. The finding that profilin can uncap actin filaments was not previously appreciated, and this newly discovered function may have important implications for filament elongation as well as depolymerization.

Actins↗

PubMed2003

Actin-based endosome and phagosome rocketing in macrophages: activation by the secretagogue antagonists lanthanum and zinc.

Although motile endocytic vesicles form actin-rich rocket tails [Merrifield et al., 1999: Nature Cell Biol 1:72-74], the mechanism of intracellular organelle locomotion remains poorly understood. We now demonstrate that bone marrow macrophages treated with lanthanum and zinc ions, well-known secretagogue antagonists, reliably exhibit vesicle motility. This treatment results in accentuated membrane ruffling and the formation of phagosomes and early endosomes that move rapidly through the cytoplasm by assembling actin filament rocket tails. Protein-specific immunolocalization demonstrated the presence of Arp2/3 complex in the polymerization zone and throughout the actin-rich tail, whereas N-WASP was most abundant in the polymerization zone. Although Arp2/3 and N-WASP play essential roles in nucleating filament assembly, other processes (i.e., elongation and filament cross-linking) are required to produce forces needed for motility. Efficient elongation was found to require zyxin, VASP, and profilin, proteins that interact by means of their ABM-1 and ABM-2 proline-rich motifs. The functional significance of these motifs was demonstrated by inhibition of vesicle motility by the motif-specific ABM-1 and ABM-2 analogues. Furthermore, lanthanum/zinc treatment also facilitated the early onset of actin-based vaccinia motility, a process that also utilizes Arp2/3 and N-WASP for nucleation and the zyxin-VASP-profilin complex for efficient elongation. Although earlier studies using cell extracts clouded the role of oligoproline sequences in activating the polymerization zone, our studies emphasize the importance of evaluating motility in living cells.

Actin-Related Protein 2↗

PubMed2003

CapG(-/-) mice have specific host defense defects that render them more susceptible than CapG(+/+) mice to Listeria monocytogenes infection but not to Salmonella enterica serovar Typhimurium infection.

Loss of the actin filament capping protein CapG has no apparent effect on the phenotype of mice maintained under sterile conditions; however, bone marrow-derived macrophages from CapG(-/-) mice exhibited distinct motility defects. We examined the ability of CapG(-/-) mice to clear two intracellular bacteria, Listeria monocytogenes and Salmonella enterica serovar Typhimurium. The 50% lethal dose of Listeria was 10-fold lower for CapG(-/-) mice than for CapG(+/+) mice (6 x 10(3) CFU for CapG(-/-) mice and 6 x 10(4) CFU for CapG(+/+) mice), while no difference was observed for Salmonella: The numbers of Listeria cells in the spleens and livers were significantly higher in CapG(-/-) mice than in CapG(+/+) mice at days 5 to 9, while the bacterial counts were identical on day 5 for Salmonella-infected mice. Microscopic analysis revealed qualitatively similar inflammatory responses in the spleens and livers of the two types of mice. Specific immunofluorescence staining analyzed by fluorescence-activated cell sorting revealed similar numbers of macrophages and dendritic cells in infected CapG(-/-) and CapG(+/+) spleens. However, analysis of bone marrow-derived macrophages revealed a 50% reduction in the rate of phagocytosis of Listeria in CapG(-/-) cells but a normal rate of phagocytosis of Salmonella: Stimulation of bone marrow-derived dendritic cells with granulocyte-macrophage colony-stimulating factor resulted in a reduction in the ruffling response of CapG(-/-) cells compared to the response of CapG(+/+) cells, and CapG(-/-) bone-marrowed derived neutrophils migrated at a mean speed that was nearly 50% lower than the mean speed of CapG(+/+) neutrophils. Our findings suggest that specific motility deficits in macrophages, dendritic cells, and neutrophils render CapG(-/-) mice more susceptible than CapG(+/+) mice to Listeria infection.

Animals↗

PubMed2002

Importance of free actin filament barbed ends for Arp2/3 complex function in platelets and fibroblasts.

We investigated the effect of actin filament barbed end uncapping on Arp23 complex function both in vivo and in vitro. Arp23 complex redistributes rapidly and uniformly to the lamellar edge of activated wild-type platelets and fibroblasts but clusters in marginal actin filament clumps in gelsolin-null cells. Treatment of gelsolin-null platelets with the negative dominant N-WASp C-terminal CA domain has no effect on their residual actin nucleation activity, placing gelsolin actin filament severing, capping, and uncapping function upstream of Arp23 complex nucleation. Actin filaments capped by gelsolin or the gelsolin homolog CapG fail to enhance Arp23 complex nucleation in vitro, but uncapping of the barbed ends of these actin filaments restores their ability to potentiate Arp23 complex nucleation. We conclude that Arp23 complex contribution to actin filament nucleation in platelets and fibroblasts importantly requires free barbed ends generated by severing and uncapping.

Actin-Related Protein 2↗

PubMed2002

A direct-transfer polymerization model explains how the multiple profilin-binding sites in the actoclampin motor promote rapid actin-based motility.

The high actin-based motility rates observed in nonmuscle cells require the per-second addition of 400-500 monomers to the barbed ends of growing actin filaments. The chief polymerization-competent species is profilin.actin.ATP (present at 5-40 microM intracellular concentrations), whereas G-actin.ATP is much less abundant ( approximately 0.1-1 microM). While earlier studies unambiguously demonstrated that profilin.actin is highly concentrated within the polymerization zone, profilin-actin localization on the motile surface cannot increase the local solution-phase concentration of polymerizable actin. To explain these high rates of actin polymerization, we present and analyze a novel polymerization model in which monomers are directly transferred to growing filament ends in the actoclampin motor. This direct-transfer polymerization mechanism endows the polymerization zone with properties unavailable to bulk-phase actin monomers, and our model also indicates why profilin is the ideal mobile carrier for actin monomers.

Actins↗

PubMed2002

Actin-based phagosome motility.

Despite abundant evidence of actin's involvement at the particle internalization stage of phagocytosis, little is known about whether phagosomes undergo the same type of actin-based motility as observed with endocytic vesicles or such intracellular pathogens as Listeria and Shigella. By employing video microscopy to follow the fate of latex bead-containing phagosomes within the cytoplasm of bone marrow macrophages, we have made the novel observation of actin-based phagosome motility. Immunofluorescence microscopy confirmed that phagosomes containing IgG-opsonized, bovine serum albumin (or BSA) -coated or uncoated latex beads all formed actin-rich rocket tails that persisted only during a brief, 1-2 min period of actin-based motility. Average speeds of actin-based phagosome motility were 0.13 +/- 0.06 microm/s for IgG-coated beads, 0.14 +/- 0.04 microm/s for BSA-coated beads, and 0.11+/- 0.03 microm/s for uncoated beads. Moreover, the speeds and motile-phase duration of each type of phagosome were comparable to the behavior of pinosomes [Merrifield et al., 1999: Nat. Cell Biol. 1:72-74.]. Determination of optimal conditions for observing and analyzing actin-based phagosome motility should facilitate future investigations of phagocytosis and phagosome maturation.

Actins↗

PubMed2002

The Chediak-Higashi protein interacts with SNARE complex and signal transduction proteins.

BACKGROUND: Chediak-Higashi syndrome (CHS) is an inherited immunodeficiency disease characterized by giant lysosomes and impaired leukocyte degranulation. CHS results from mutations in the lysosomal trafficking regulator (LYST) gene, which encodes a 425-kD cytoplasmic protein of unknown function. The goal of this study was to identify proteins that interact with LYST as a first step in understanding how LYST modulates lysosomal exocytosis. MATERIALS AND METHODS: Fourteen cDNA fragments, covering the entire coding domain of LYST, were used as baits to screen five human cDNA libraries by a yeast two-hybrid method, modified to allow screening in the activation and the binding domain, three selectable markers, and more stringent confirmation procedures. Five of the interactions were confirmed by an in vitro binding assay. RESULTS: Twenty-one proteins that interact with LYST were identified in yeast two-hybrid screens. Four interactions, confirmed directly, were with proteins important in vesicular transport and signal transduction (the SNARE-complex protein HRS, 14-3-3, and casein kinase II). CONCLUSIONS: On the basis of protein interactions, LYST appears to function as an adapter protein that may juxtapose proteins that mediate intracellular membrane fusion reactions. The pathologic manifestations observed in CHS patients and in mice with the homologous mutation beige suggest that understanding the role of LYST may be relevant to the treatment of not only CHS but also of diseases such as asthma, urticaria, and lupus, as well as to the molecular dissection of the CHS-associated cancer predisposition.

14-3-3 Proteins↗